We have used the proteasome inhibitor bortezomib to sensitize human tumor cell lines to apoptosis mediated by death ligands, in particular the protein TRAIL. Also we have performed studies with antibodies that act as agonists to the TRAIL death receptors. The agonist antibodies may offer specific advantages for cancer therapy. There are 4 well-defined TRAIL receptors in humans, yet only 2 of them death receptor 4 (DR4) and death receptor 5 (DR5) send the apoptotic death signal. Therefore specific agonist antibodies to these receptors may be more selective than the TRAIL protein itself that can bind to all of the receptors. In addition antibodies tend to have a much longer half-life that the TRAIL protein in vivo, so their effects may be more potent and longer lasting. Bortezomib, a novel anticancer agent that inhibits the proteasome complex of enzymes recently was approved by the FDA for the therapy of multiple myeloma as a single agent. In mouse models of renal cancer and breast cancer we showed that agonist antibodies to the mouse TRAIL death receptor DR5 had significantly enhanced pro-apoptotic activity in vitro in the presence of the proteasome inhibitor bortezomib. More importantly, this combination of agents had an improved therapeutic effect over either agent alone in experimental lung metastases models of both the renal and breast carcinomas in vivo. In addition the therapeutic combination did not cause any obvious toxicity in the mice on examination of a large number of organs. This was the first demonstration, to our knowledge, that this combination could have therapeutic anticancer efficacy in vivo in the absence of significant toxicity. We investigated the molecular basis of this sensitization in more detail. Cell death by apoptosis in response to the death ligands can proceed by 2 pathways. These are the extrinsic pathway, where the signaling from the death ligand death receptors is strong and directly signals cell death by setting off a cascade of enzymes. Alternatively for the intrinsic pathway, signaling from the death receptors is weaker and subsequent damage to the mitochondria of the cell is necessary for the cell death to occur. Using human renal carcinoma cells that are either sensitized or resistant to the effects of bortezomib on TRAIL-driven apoptosis, we concluded that the major effects of bortzomib were on amplifying the extrinsic apoptotic pathway. However exactly how a proteasome inhibitor amplifies this signaling at the molecular level is unclear and is under further investigation. Furthermore, in collaboration with the Molecular Targeting Development Program (MTDP) of the NCI we have also established a high-throughput screen to identify novel agents that can sensitize human cancer cells to TRAIL-mediated apoptosis. Over 50,000 pure synthetic compounds or natural products were screened and 18 compounds were identified for further study, 14 pure products and 4 synthetic compounds. Three of the compounds have been studied in more detail and none are proteasome inhibitors. We have identified two families of natural products, the cucurbitacins and the withanolides, where some family members sensitize tumor cells to TRAIL apoptosis whereas others do not. We recently published a manuscript on TRAIL sensitization by the cucurbitacins. Since the cucurbitacins are quite toxic, most of our current studies concern the withanolides. We have identified withanolide E as an extremely good sensitizer of various cancer cells to TRAIL-mediated apoptosis. It appears to sensitize the cancer cells by rapidly reducing the levels of the anti-apoptotic protein cFLIP. This is due to destabilization of the protein resulting in its degradation. The mechanism may involve binding of withanolide E to the chaperone protein HSP90 thus blocking the stabilization of cFLIP. In contrast to other members of the withanolide family, withanolide E seems to selectively reduce cFLIP, and a few other select HSP90 client proteins.This may be due to its interaction site on HSP90, and this is under further investigation. Interestingly withanolide E can also sensitize cancer cells to the apoptotic effects of other TNF family death ligands. We have also recently marked a cancer cell population with green fluorescent protein (GFP) that has many characterisitics of a cancer stem cell population. This allows for isolation of these cells based on their GFP expression. The sensitivity of these cancer cell subpopulations to apoptosis by various death ligands in combination with withanolide E will be examined in the future